Normal human epidermal keratinocytes grown in a serum-free keratinocyte growth medium were evaluated as an in vitro alternative to the Draize ocular irritation test. The cells were cultured in 96-we11 tissue culture plates and treated with test agents. Cytotoxicity of the agent was estimated by the uptake of neutral red by viable cells. Fifty-one chemicals which are used in cosmetics were employed as test agents. The correlation coefficient between in vivo Draize score and in vitro test with normal human epidermal keratinocytes was 0.83. In addition, the method using normal human epidermal keratinocytes was more sensitive to a given concentration of test agents than the colony forming assay using rabbit cornea primary cells. These results suggested that the cytotoxicity test in vitro using normal human epidermal keratinocytes is useful as an alternative to the ocular irritating test.